UK Standards for Microbiology Investigations
Identification of Staphylococcus species, Micrococcus
species and Rothia species
Issued by the Standards Unit, Microbiology Services, PHE
Bacteriology – Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 1 of 32
© Crown copyright 2014
Identification of Staphylococcus species, Micrococcus species and Rothia species
Acknowledgments
UK Standards for Microbiology Investigations (SMIs) are developed under the
auspices of Public Health England (PHE) working in partnership with the National
Health Service (NHS), Public Health Wales and with the professional organisations
whose logos are displayed below and listed on the website https://www.gov.uk/ukstandards-for-microbiology-investigations-smi-quality-and-consistency-in-clinicallaboratories. SMIs are developed, reviewed and revised by various working groups
which are overseen by a steering committee (see
https://www.gov.uk/government/groups/standards-for-microbiology-investigationssteering-committee).
The contributions of many individuals in clinical, specialist and reference laboratories
who have provided information and comments during the development of this
document are acknowledged. We are grateful to the Medical Editors for editing the
medical content.
For further information please contact us at:
Standards Unit
Microbiology Services
Public Health England
61 Colindale Avenue
London NW9 5EQ
E-mail: standards@phe.gov.uk
Website: https://www.gov.uk/uk-standards-for-microbiology-investigations-smi-qualityand-consistency-in-clinical-laboratories
UK Standards for Microbiology Investigations are produced in association with:
Logos correct at time of publishing.
Bacteriology – Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 2 of 32
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
Identification of Staphylococcus species, Micrococcus species and Rothia species
Contents
ACKNOWLEDGMENTS .......................................................................................................... 2
AMENDMENT TABLE ............................................................................................................. 4
UK STANDARDS FOR MICROBIOLOGY INVESTIGATIONS: SCOPE AND PURPOSE ....... 6
SCOPE OF DOCUMENT ......................................................................................................... 9
INTRODUCTION ..................................................................................................................... 9
TECHNICAL INFORMATION/LIMITATIONS ......................................................................... 15
1
SAFETY CONSIDERATIONS .................................................................................... 17
2
TARGET ORGANISMS .............................................................................................. 17
3
IDENTIFICATION ....................................................................................................... 18
4
IDENTIFICATION OF STAPHYLOCOCCUS SPECIES, MICROCOCCUS SPECIES
AND ROTHIA SPECIES ............................................................................................. 23
5
REPORTING .............................................................................................................. 24
6
REFERRALS.............................................................................................................. 25
7
NOTIFICATION TO PHE OR EQUIVALENT IN THE DEVOLVED
ADMINISTRATIONS .................................................................................................. 25
REFERENCES ...................................................................................................................... 27
Bacteriology – Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 3 of 32
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
Identification of Staphylococcus species, Micrococcus species and Rothia species
Amendment Table
Each SMI method has an individual record of amendments. The current amendments
are listed on this page. The amendment history is available from
standards@phe.gov.uk.
New or revised documents should be controlled within the laboratory in accordance
with the local quality management system.
Amendment No/Date.
5/12.11.14
Issue no. discarded.
2.3
Insert Issue no.
3
Section(s) involved
Amendment
Whole document.
Hyperlinks updated to gov.uk.
Page 2.
Updated logos added.
Whole document.
Document presented in a new format.
Scope of document.
The scope has been updated to include webpage
links for B 29 and ID 4 documents.
The taxonomy of Staphylococcus, Micrococcus
and Rothia species has been updated.
Introduction.
More information has been added to the
Characteristics section. The medically important
species are mentioned and their characteristics
described.
Use of up-to-date references.
Section on Principles of Identification has been
updated to reflect rapid methods used for
identification.
Technical
Information/Limitations.
Addition of information regarding agar media,
coagulase test and common issues with S. aureus
has been described and referenced.
Target Organisms.
The section on the Target organisms has been
updated and presented clearly. References have
been updated.
Minor amendments have been made to 3.1 and
3.2.
Identification.
3.3 and 3.4 have been updated to reflect
standards in practice.
Subsection 3.5 has been updated to include the
Rapid Molecular Methods.
Bacteriology – Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 4 of 32
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
Identification of Staphylococcus species, Micrococcus species and Rothia species
Identification Flowchart.
Modification of flowchart for identification of
species has been made for easy guidance.
Reporting.
Subsection 5.1 - 5.4 has been updated to reflect
reporting practice.
Referral.
The address of the reference laboratory has been
updated.
References.
Some references updated.
Bacteriology – Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 5 of 32
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
Identification of Staphylococcus species, Micrococcus species and Rothia species
UK Standards for Microbiology Investigations#:
Scope and Purpose
Users of SMIs
•
SMIs are primarily intended as a general resource for practising professionals
operating in the field of laboratory medicine and infection specialties in the UK.
•
SMIs provide clinicians with information about the available test repertoire and
the standard of laboratory services they should expect for the investigation of
infection in their patients, as well as providing information that aids the
electronic ordering of appropriate tests.
•
SMIs provide commissioners of healthcare services with the appropriateness
and standard of microbiology investigations they should be seeking as part of
the clinical and public health care package for their population.
Background to SMIs
SMIs comprise a collection of recommended algorithms and procedures covering all
stages of the investigative process in microbiology from the pre-analytical (clinical
syndrome) stage to the analytical (laboratory testing) and post analytical (result
interpretation and reporting) stages.
Syndromic algorithms are supported by more detailed documents containing advice
on the investigation of specific diseases and infections. Guidance notes cover the
clinical background, differential diagnosis, and appropriate investigation of particular
clinical conditions. Quality guidance notes describe laboratory processes which
underpin quality, for example assay validation.
Standardisation of the diagnostic process through the application of SMIs helps to
assure the equivalence of investigation strategies in different laboratories across the
UK and is essential for public health surveillance, research and development activities.
Equal Partnership Working
SMIs are developed in equal partnership with PHE, NHS, Royal College of
Pathologists and professional societies.
The list of participating societies may be found at https://www.gov.uk/uk-standards-formicrobiology-investigations-smi-quality-and-consistency-in-clinical-laboratories.
Inclusion of a logo in an SMI indicates participation of the society in equal partnership
and support for the objectives and process of preparing SMIs. Nominees of
professional societies are members of the Steering Committee and Working Groups
which develop SMIs. The views of nominees cannot be rigorously representative of
the members of their nominating organisations nor the corporate views of their
organisations. Nominees act as a conduit for two way reporting and dialogue.
Representative views are sought through the consultation process.
SMIs are developed, reviewed and updated through a wide consultation process.
#
Microbiology is used as a generic term to include the two GMC-recognised specialties of Medical Microbiology (which includes
Bacteriology, Mycology and Parasitology) and Medical Virology.
Bacteriology – Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 6 of 32
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
Identification of Staphylococcus species, Micrococcus species and Rothia species
Quality Assurance
NICE has accredited the process used by the SMI Working Groups to produce SMIs.
The accreditation is applicable to all guidance produced since October 2009. The
process for the development of SMIs is certified to ISO 9001:2008.
SMIs represent a good standard of practice to which all clinical and public health
microbiology laboratories in the UK are expected to work. SMIs are NICE accredited
and represent neither minimum standards of practice nor the highest level of complex
laboratory investigation possible. In using SMIs, laboratories should take account of
local requirements and undertake additional investigations where appropriate. SMIs
help laboratories to meet accreditation requirements by promoting high quality
practices which are auditable. SMIs also provide a reference point for method
development.
The performance of SMIs depends on competent staff and appropriate quality
reagents and equipment. Laboratories should ensure that all commercial and in-house
tests have been validated and shown to be fit for purpose. Laboratories should
participate in external quality assessment schemes and undertake relevant internal
quality control procedures.
Patient and Public Involvement
The SMI Working Groups are committed to patient and public involvement in the
development of SMIs. By involving the public, health professionals, scientists and
voluntary organisations the resulting SMI will be robust and meet the needs of the
user. An opportunity is given to members of the public to contribute to consultations
through our open access website.
Information Governance and Equality
PHE is a Caldicott compliant organisation. It seeks to take every possible precaution
to prevent unauthorised disclosure of patient details and to ensure that patient-related
records are kept under secure conditions.
The development of SMIs are subject to PHE Equality objectives
https://www.gov.uk/government/organisations/public-health-england/about/equalityand-diversity. The SMI Working Groups are committed to achieving the equality
objectives by effective consultation with members of the public, partners, stakeholders
and specialist interest groups.
Legal Statement
Whilst every care has been taken in the preparation of SMIs, PHE and any supporting
organisation, shall, to the greatest extent possible under any applicable law, exclude
liability for all losses, costs, claims, damages or expenses arising out of or connected
with the use of an SMI or any information contained therein. If alterations are made to
an SMI, it must be made clear where and by whom such changes have been made.
The evidence base and microbial taxonomy for the SMI is as complete as possible at
the time of issue. Any omissions and new material will be considered at the next
review. These standards can only be superseded by revisions of the standard,
legislative action, or by NICE accredited guidance.
SMIs are Crown copyright which should be acknowledged where appropriate.
Bacteriology – Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 7 of 32
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
Identification of Staphylococcus species, Micrococcus species and Rothia species
Suggested Citation for this Document
Public Health England. (2014). Identification of Staphylococcus species, Micrococcus
species and Rothia species . UK Standards for Microbiology Investigations. ID 7 Issue
3. https://www.gov.uk/uk-standards-for-microbiology-investigations-smi-quality-andconsistency-in-clinical-laboratories
Bacteriology – Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 8 of 32
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
Identification of Staphylococcus species, Micrococcus species and Rothia species
Scope of Document
This SMI describes the identification of Staphylococcus species, Micrococcus species
and Rothia species. Details on MRSA screening can be found in B 29 - Investigation
of Specimens for Screening MRSA.
For the identification of catalase negative Gram positive cocci, see ID 4 - Identification
of Streptococcus species, Enterococcus species and Morphologically Similar
Organisms.
This SMI should be used in conjunction with other SMIs.
Introduction
Taxonomy
Taxonomically, the genus Staphylococcus is in the bacterial family
Staphylococcaceae, which includes five lesser known genera, Gemella,
Jeotgalicoccus, Macrococcus, Nosocomiicoccus and Salinicoccus. There are currently
47 recognised species of staphylococci and 21 subspecies most of which are found
only in lower mammals1. The staphylococci most frequently associated with human
infection are S. aureus, S. epidermidis and S. saprophyticus. Other Staphylococcus
species may also be associated with human infection2.
The genus Micrococcus belongs to the bacterial family Micrococcaceae which
currently contains 16 species. These have been isolated from human skin, animal and
dairy products as well as environment (water, dust and soil)3. Some of these species
have been re-classified to other genera. Former members of the genus Micrococcus,
now assigned to other genera, include Arthrobacter agilis, Nesterenkonia halobia,
Kocuria kristinae, K. rosea, K. varians, Kytococcus sedentarius, and Dermacoccus
nishinomiyaensis. The Micrococcus species that are associated with infections are
Micrococcus luteus and Micrococcus lylae.
The genus Rothia belonged to the bacterial family Actinomycetaceae as described by
Georg and Brown in 1967 but more recent molecular studies placed the genus in the
family Micrococcaceae, suborder Micrococcineae, order Actinomycetales, subclass
Actinobacteridae and class Actinobacteria. It is therefore in the same family as the
genera Micrococcus, Arthrobacter, Kocuria, Nesterenkonia, Renibacterium and
Stomatococcus, all of which show characteristic signature nucleotides in their 16S
rDNA sequences4. There are currently 6 species, Rothia dentocariosa and Rothia
mucilaginosa are the only two which have been known to cause infections in humans5.
Characteristics
Staphylococcus species are Gram positive, non-motile, non-sporing cocci of varying
size occurring singly, in pairs and in irregular clusters. Colonies are opaque and may
be white or cream and occasionally yellow or orange. The optimum growth
temperature is 30°C-37°C. They are facultative anaerobes and have a fermentative
metabolism. Staphylococcus species are usually catalase positive and are also
oxidase negative with the exception of the S. sciuri group (S. sciuri, S. lentus and
S. vitulinus), S. fleuretti and the Macrococcus group to which S. caseolyticus has been
assigned2,6,7. This is also a distinguishing factor from the genus streptococci, which
are catalase negative, and have a different cell wall composition to staphylococci.
Bacteriology – Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 9 of 32
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
Identification of Staphylococcus species, Micrococcus species and Rothia species
Nitrate is often reduced to nitrite. Some species are susceptible to lysis by lysostaphin
but not lysozyme and are able to grow in 6.5% sodium chloride. Some species
produce extracellular toxins. Staphylococci may be identified by the production of
deoxyribonuclease (DNase) and/or a heat-stable DNase (thermostable nuclease)8.
Coagulase positive staphylococci
Staphylococcus aureus
S. aureus are cocci that form irregular grape-like clusters. They are non-motile, nonsporing and catalase positive. They grow rapidly and abundantly under aerobic
conditions. On blood agar, they appear as glistening, smooth, entire, raised,
translucent colonies that often have a golden pigment. The colonies are 2-3mm in
diameter after 24hr incubation and most strains show β-haemolysis surrounding the
colonies.
There are currently 2 subspecies of S. aureus; these are S. aureus subspecies aureus
and S. aureus subspecies anaerobius.
S. aureus subspecies aureus is commonly isolated from human clinical specimens.
All strains are able to grow on thioglycolate medium within 24hr. Most strains produce
a wide zone of strong haemolysis within 24 to 36hr. They show positivity for Dglucose, D-fructose, D-mannose, D-maltose, D-lactose, D-trehalose, D-mannitol,
sucrose, N-acetyl-glucosamine, D-celiobiose, and D-turanose; meanwhile, no acid
production was demonstrated by utilization of D-ribose, xylitol, xylose, D-melibiose,
raffinose, L-arabinose, and a-methyl-D-glucoside. They are also positive for catalase,
coagulase, and benzidine reactions and are capable of nitrate reduction and
acetylmethylcarbinol (acetoin) production. Results for DNase, clumping factor, urease,
arginine dihydrolase, pyrrolidonyl arylamidase, leucine arylamidase,
β-N-acetylglucosaminidase, α-chymotrypsin, α-glucosidase, β-glucosidase, alkaline
phosphatase, esterase C-4 and C-8, lipase (C-14), phosphatase acid, and
naphthol- AS-BI-phosphohydrolase are positive.
There is no production of oxidase, α-galactosidase, β- glucoronidase, β-galactosidase,
valine arylamidase, cystine arylamidase, arginine arylamidase, trypsin, ornithine
decarboxylase, α-mannosidase, and α-fucosidase. All strains are resistant to
novobiocin9.
S. aureus subspecies anaerobius is rarely isolated from clinical specimens. They
are 0.8 to 1.0µm in diameter and occur singly, in pairs, and predominantly in irregular
clusters. On the primary isolation medium, growth is obtained only in media that are
supplemented with blood, serum, or egg yolk and incubated microaerobically or
anaerobically. Colonies on blood agar after 2 days of incubation are very small (1 to
3mm in diameter), low convex, circular, entire, smooth, glistening, and opaque.
Pigment is not produced. Luxuriant growth is obtained on Dorset egg medium, with
colony diameters of 4 to 6mm. The strains produce unevenly disseminated growth on
brain heart infusion agar after 3 days of microaerophilic incubation. They grow as
dwarf colonies, among which a few colonies of normal size are observed10.
It grows poorly aerobically and growth may be CO2 dependent. It is slide coagulase
negative and thermonuclease negative and may be catalase negative. Strains may be
identified by better growth anaerobically and they may give a positive coagulase test
result. However, because growth may be poor, the coagulase result may be negative
and suspected isolates should be referred to the Reference Laboratory.
Bacteriology – Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 10 of 32
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
Identification of Staphylococcus species, Micrococcus species and Rothia species
Staphylococcus aureus may be associated with severe infection and it is important to
distinguish it from the opportunistic coagulase negative staphylococci. In routine
laboratory practice, the production of coagulase is frequently used as the sole criterion
to distinguish S. aureus from other staphylococci. It is also important to note that
coagulase negative strains of S. aureus have been reported11.
Other coagulase positive staphylococcal species such as S. hyicus, S. schleiferi
subspecies coagulans, S. pseudintermedius or S. intermedius may be coagulase
positive but have been found only occasionally to be associated with human infection
or carriage12,13. The production of coagulase and thermostable nuclease by these
staphylococci may lead to their misidentification as S. aureus. Staphylococcus delphini
is coagulase positive and thermostable nuclease positive (rarely isolated from
humans).
Carbon dioxide dependent strains of S. aureus can be recovered from clinical
material14. The significance of these strains in the laboratory is that they pose a
significant technical problem when performing antibiotic susceptibility testing as they
fail to grow in air. Therefore susceptibility testing should be performed in a CO2
enriched atmosphere. These strains although referred to as dwarf strains in the past
should not be confused with the slow-growing small colony variants (SCV’s) of
S. aureus which have decreased metabolism and a defective electron transport
system and are auxotrophic for substrates such as haemin, menadione, thiamine or
thymidine15. Such strains are meticillin resistant and have an intrinsic resistance to
aminoglycoside antibiotics such as gentamicin and are most frequently identified in
patients with chronic or persistent infections16.
Multi resistance to antibiotics has most often been associated with meticillin resistant
strains16.
Staphylococcus aureus produces virulence factors such as protein A, capsular
polysaccharides and α toxin. Some strains of S. aureus produce toxic shock
syndrome 1 toxin (TSST-1), Panton-Valentine Leucocidin or other toxins.
Coagulase negative staphylococci (CoNS)17
Coagulase negative staphylococci (CoNS) are normal commensals of the skin,
anterior nares, and ear canals of humans. They have long been considered as nonpathogenic, and were rarely reported to cause severe infections. However, as a result
of the combination of increased use of intravascular devices and an increase in the
number of hospitalized immunocompromised patients, CoNS have emerged as a
major cause of nosocomial bloodstream infections.
They are opportunistic pathogens which lack many of the virulence factors associated
with S. aureus. There are more than 30 species of CoNS. The taxonomy of these
coagulase negative staphylococci (CoNS) fall into clusters based on 16s rRNA
sequences18.
S. epidermidis and S. saprophyticus are the species most often associated with
infection but Staphylococcus capitis, Staphylococcus cohnii, Staphylococcus
haemolyticus, Staphylococcus hominis, Staphylococcus lugdunensis, Staphylococcus
sciuri, Staphylococcus schleiferi subspecies schleiferi, Staphylococcus simulans,
Staphylococcus saccharolyticus (previously known as Peptococcus saccharolyticus)
and Staphylococcus warneri have also been implicated19,20. Many of these species are
also thermostable nuclease negative. S. lugdunensis is coagulase negative but some
strains may be positive for the slide coagulase test or clumping factor 21.
Bacteriology – Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 11 of 32
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
Identification of Staphylococcus species, Micrococcus species and Rothia species
Multi resistance to antibiotics also occurs in some strains of S. epidermidis which are
thermostable nuclease negative. S. saprophyticus, S. cohnii and S. sciuri groups are
generally novobiocin resistant as is S. hominis subsp. novobiosepticus22.
Staphylococcus pasteuri can be phenotypically distinguished from all of the other
novobiocin-susceptible staphylococci except S. warneri, from which it can only be
differentiated by genotyping23.
Staphylococcus epidermidis
S. epidermidis are approximately 0.5 to 1.5µm in diameter and arranged in grape-like
clusters. They are facultative anaerobes that can grow by aerobic respiration or by
fermentation. Some strains may not ferment.
It forms greyish-white, raised, circular, smooth, glistening, and translucent to slightly
opaque, cohesive colonies approximately 1–2mm in diameter after overnight
incubation, and is non-haemolytic on blood agar. They grow well at NaCl
concentrations up to 7.5%, poorly at 10% and fail to grow at 15%. They are positive
for catalase, urease and exhibit a weak positive reaction for the nitrate reduction test.
They are negative for coagulase, oxidase and gelatin hydrolysis tests. They utilize
glucose, fructose, sucrose, and lactose to form acid products aerobically. In the
presence of lactose, they will also produce gas.
They are either susceptible or slightly resistant to lysostaphin and are resistant to
lysozyme.
S. epidermidis is sensitive to novobiocin, and this test distinguishes it from
Staphylococcus saprophyticus, which is coagulase negative, as well, but novobiocin
resistant24.
Staphylococcus saprophyticus
They are positive for catalase and urease tests while they are negative for motility,
coagulase, nitrate reduction and oxidase tests. They utilize fructose, maltose, sucrose
and trehalose to form acid products. They grow well on 10% NaCl agar, but only 1189% strains tolerate 15% NaCl. Colonies appear as raised to slightly convex, circular,
usually entire, 4.0 to 9.0mm in diameter, smooth, glistening, and usually opaque.
Colony pigment is variable; however, most strains are not pigmented or might have a
slight yellow tint which increases in intensity with age.
Two subspecies for S. saprophyticus exist: S. saprophyticus subsp. bovis and
S. saprophyticus subsp. saprophyticus, the latter is more commonly found in human
UTIs. S. saprophyticus subsp. saprophyticus is distinguished by its being nitrate
reductase and pyrrolidonyl arylamidase negative while S. saprophyticus subsp. bovis
is nitrate reductase and pyrolidonyl arymamidase positive25.
S. saprophyticus is resistant to the antibiotic novobiocin, a characteristic that is used in
laboratory identification to distinguish it from S. epidermidis, which is also coagulase
negative but novobiocin sensitive22.
Micrococcus species
Micrococcus species are strictly aerobic Gram positive cocci arranged in tetrads or
irregular clusters, not in chains and cells range from 0.5 to 3µm in diameter. They are
seldom motile and are non-sporing. They are also catalase positive and often oxidase
positive, although weakly. Micrococci may be distinguished from staphylococci by a
modified oxidase test26,27. Their colonies are usually pigmented in shades of yellow or
red and grow on simple media. The optimum growth temperature is 25-37°C. They
Bacteriology – Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 12 of 32
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
Identification of Staphylococcus species, Micrococcus species and Rothia species
have a respiratory metabolism, often producing little or no acid from carbohydrates
and are usually halotolerant, growing in 5% NaCl. They contain cytochromes and are
resistant to lysostaphin8.
They are generally considered harmless saprophytes that inhabit or contaminate the
skin, mucosa, and also the oropharynx; however they can be opportunistic pathogens
in certain immunocompromised patients22.
There are currently 9 species of Micrococcus and 2 have been known to cause
infections in humans - Micrococcus lylae and Micrococcus luteus3.
Micrococcus lylae
They are mostly arranged in tetrads. They are positive for catalase and oxidase and
negative for urease. They grow in circular, entire, convex and usually not pigmented or
cream white colonies having diameters of approximately 4mm after 2-3 days on plate
at 37°C. They assimilate D- Maltose, D- Trehalose, Maltitol, acetate, citrate,
d-Glucose, sucrose, d-fructose, d, fumarate, dl-3-hydroxybutyrate, dl-lactate, pyruvate,
l-aspartate, l-histidine, l-leucine, 3-hydroxybenzoate and 4-hydroxybenzoate and also
hydrolyse l-proline pNA, Tween 20 and Tween 8028.
M. lylae can be distinguished from the closely related species M. luteus by lysozyme
susceptibility, genetic compatibility, and the type of cell-wall peptidoglycan. There are
also some differences between these species in the parameters of pigmentation,
nitrogen requirements, nitrate reduction and acid from maltose and sucrose.
It has been isolated from human skin.
Micrococcus luteus
They are mostly arranged in tetrads. They are positive for catalase and oxidase. They
grow in circular, entire, convex and creamy yellow pigmented colonies having
diameters of approximately 4mm after 2-3 days at 37°C. Several uncommon strains
produce raised colonies with translucent, depressed centres. Colony pigmentation
varies considerably but are usually different shades of yellow or cream-white29.
Growth or weak growth is observed at 45°C, at pH 10 and in the presence of 10%
NaCl; no growth is observed in the presence of 15% NaCl. D-Glucose, sucrose and
D-mannose are assimilated while L-proline pNA, and Tween 20 are hydrolysed.
There are 3 biovars of M. luteus and they possess quite diverse chemotaxonomic
features with respect to their menaquinone systems, cell-wall compositions and
Fourier transform-infrared (FT-IR) spectroscopy (FT-IR) patterns, as well as
biochemical properties. The recognition of three different biovars within the species
M. luteus has the advantage that the three groups can be differentiated without
nomenclatural changes having to be introduced28.
It has been isolated from human skin.
Rothia species
Rothia species are Gram positive cocci with a variable microscopic morphology. Their
cells occur singly, in pairs, in clusters or in chains. They are weakly catalase positive
and weakly proteolytic. Rothia species are positive for nitrate and nitrite reduction,
liquefaction of gelatin and fermentation of sugars with the production of acid; while
negative for motility, urease and indole. Colonies on agar surface may appear
branched which rapidly fragment into bacillary or coccoid forms, resembling
Bacteriology – Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 13 of 32
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
Identification of Staphylococcus species, Micrococcus species and Rothia species
Actinomyces or Nocardia species30. They exhibit good growth under aerobic or
microaerophilic conditions, but poor or no growth anaerobically.
Rothia species are susceptible to penicillin but because rare isolates may be resistant,
susceptibility testing should be performed.
There are currently 7 species of Rothia and 2 have been known to cause infections in
humans - Rothia dentocariosa and Rothia mucilaginosa5.
Rothia dentocariosa
R. dentocariosa cells occur singly, in pairs, in clusters or in chains. Colonial
pleomorphism can also be observed. Microscopically, the morphology varies from
coccoid to diphtheroid (with clavate ends) to filamentous. In broth cultures, cells may
be coccoid, which distinguishes them from Actinomyces species and appears in
filamentous forms on plates, but mixtures may appear in any culture4. They may show
rudimentary branching and loss of the Gram positive appearance in ageing cultures.
R. dentocariosa grows faster under aerobic than under anaerobic conditions, and
does not need CO2 or lipids for growth. It grows well on simple media (except
Sabouraud dextrose agar) and colonies may be creamy, dry, crumbly or mucoid, nonhaemolytic and may adhere to the agar surface. They are non-motile, catalase
positive and ferment carbohydrates with the end-products being lactic and acetic
acid31.
Catalase negative strains of R. dentocariosa have been reported and this will be more
difficult to recognise with traditional tests, since they may mimic the rare
Bifidobacterium strains that are able to grow aerobically, as well as Actinomyces and
Arcanobacterium species, Propionibacterium propionicum and catalase negative
Listeria strains4.
R. dentocariosa is distinct from Dermabacter species in that it is nitrate and
pyrazinamidase positive.
Rothia mucilaginosa (was previously known as Stomatococcus mucilaginosus,
Micrococcus mucilaginosus or Staphylococcus salivarius32,33.
This is found in clusters. Cells display variable catalase reactions ranging from
negative to weakly positive to strongly positive, oxidase negative, and exhibit
facultatively anaerobic metabolism. They are able to use glucose fermentatively.
Optimum growth temperature is 30-37°C. Their white to greyish non-haemolytic
colonies may be mucoid, rubbery, or sticky in consistency and adherent to agar due to
the mucilagenous capsular material produced. The inability to grow in the presence of
5% NaCl distinguishes R. mucilaginosa from members of the genera Staphylococcus
and Micrococcus34.
It is isolated primarily from mouth and respiratory tract of humans, and is capable of
growth and producing diseases like endocarditis and meningitis in mammals.
Principles of Identification
Presumptive staphylococci need to be quickly differentiated into two groups:
•
Probable S. aureus - a potential pathogen when isolated from most sites
•
Other staphylococci - usually not significant in skin and superficial wound swab
sites, but a possible pathogen in some circumstances
Bacteriology – Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 14 of 32
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
Identification of Staphylococcus species, Micrococcus species and Rothia species
Staphylococcus aureus has traditionally been identified by tube coagulase tests that
detect staphylocoagulase or "free coagulase". However, detection of surface proteins
such as clumping factor (slide coagulase test) and/or protein A (commercial latex
tests) may be used for rapid identification. Inclusion of latex particles sensitized with
antibodies against specific capsular antigens has enabled commercial manufacturer’s
to improve the sensitivity of latex tests to detect atypical strains of S. aureus and
MRSA that fail to express the major characteristics listed above35. Positive results or
suspected erroneous slide tests may be confirmed by a tube coagulase test.
Full molecular identification using for example, MALDI-TOF MS can be used to identify
CoNS isolates to species level.
Typing and differentiation between strains of S. aureus can be achieved using a range
of molecular techniques eg spa typing, Pulsed Field Gel Electrophoresis (PFGE),
Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA), Multi-locus
sequence typing (MLST), Microarrays, Next Generation Sequencing, etc36.
Technical Information/Limitations
Agar Media
The use of conventional media such as blood agar has the advantage that they may
also be useful for the simultaneous isolation of other pathogens such as streptococci.
The disadvantage of such media is that confirmatory tests are necessary to
differentiate S. aureus from other staphylococci35. Performing such tests on all
colonies resembling staphylococci can be time-consuming and labour intensive.
The use of chromogenic media, if sufficiently sensitive and specific, can potentially
reduce the number of confirmatory tests and achieve isolation and presumptive
identification in a single step. Another advantage is that they require fewer reagents
for confirmation of suspect colonies of S. aureus and hence may be cost effective37,38.
Chromogenic media for S. aureus may be supplemented with appropriate
antimicrobials (eg oxacillin or cefoxitin) for the detection of MRSA39.
Note: Chromogenic media are affected by direct light and plates should be stored in
the dark and not left in the light long before or after inoculation.
Coagulase test
S. aureus is differentiated from other staphylococci by the coagulase test. However it
is now known that not all S. aureus are coagulase positive and not all coagulase
positive staphylococci are S. aureus40.
S. lugdunensis is coagulase negative but some strains may be slide coagulase or
clumping factor positive.
For the tube coagulase test, citrate-utilizing organisms such as Enterococcus faecalis,
Pseudomonas species, Serratia marcescens, and strains of Streptococcus will clot
citrated plasma41.
S. hyicus, S. intermedius, S. pseudintermedius and S. schleiferi may be tube
coagulase positive.
Bacteriology – Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 15 of 32
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
Identification of Staphylococcus species, Micrococcus species and Rothia species
Common issues with S. aureus
Yeasts can be misidentified as coagulase negative staphylococci on the basis of
colony morphology and a negative slide agglutination test. Speciation of staphylococci
should be considered for isolates from sterile sites and blood cultures to avoid missing
S. aureus, S. lugdunensis or yeasts.
Staphylococcus sciuri can give positive results with DNA and Staph aureus latex tests
and can have the mecA gene and therefore grow on chromogenic MRSA medium with
a blue green pigment. On blood agar, they appear as large yellow colonies resembling
S. aureus. It is easily distinguished from other staphylococcci as it is oxidase positive.
Other non S. aureus species such as S. intermedius could also be misidentified as
MRSA/MSSA.
Bacteriology – Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 16 of 32
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
Identification of Staphylococcus species, Micrococcus species and Rothia species
1
Safety Considerations42-58
Refer to current guidance on the safe handling of all organisms documented in this
SMI.
Laboratory procedures that give rise to infectious aerosols must be conducted in a
microbiological safety cabinet50.
The above guidance should be supplemented with local COSHH and risk
assessments.
Compliance with postal and transport regulations is essential.
2
Target Organisms
Staphylococcus species reported to have caused human infections13,21,22,59-64
S. aureus group - Staphylococcus aureus subsp aureus, Staphylococcus aureus
subsp anaerobius
S. epidermidis group - Staphylococcus epidermidis, Staphylococcus capitis subsp
capitis, Staphylococcus capitis subsp urealyticus, Staphylococcus caprae,
Staphylococcus saccharolyticus
S. saprophyticus group - Staphylococcus saprophyticus subsp saprophyticus,
Staphylococcus cohnii subsp cohnii, Staphylococcus cohnii subsp urealyticus,
Staphylococcus xylosus
S. hyicus-intermedius group - Staphylococcus hyicus, Staphylococcus intermedius,
Staphylococcus pseudintermedius, Staphylococcus schleiferi subsp coagulans,
Staphylococcus schleiferi subsp schleiferi
S. simulans group - Staphylococcus simulans
S. haemolyticus group - Staphylococcus hominis subsp hominis, Staphylococcus
hominis subsp novobiosepticus, Staphylococcus haemolyticus
S. lugdunensis group - Staphylococcus lugdunensis
S. warneri group - Staphylococcus warneri, Staphylococcus pasteuri
S. auricularis group - Staphylococcus auricularis
S. carnosus group - Staphylococcus massiliensis, Staphylococcus pettenkoferi
S. sciuri group - Staphylococcus sciuri subsp sciuri, Staphylococcus sciuri subsp
rodentium,
Staphylococcus sciuri subsp carnaticus, Staphylococcus lentus, Staphylococcus
vitulinus
Other species reported to have caused human infections- Micrococcus luteus,
Micrococcus lylae, Micrococcus mortus (not officially recognised), Rothia
mucilaginosa, Rothia dentocariosa
Bacteriology – Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 17 of 32
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
Identification of Staphylococcus species, Micrococcus species and Rothia species
3
Identification
3.1
Microscopic Appearance
Gram stain (TP 39 - Staining Procedures)
Gram positive cocci occurring singly, in pairs, tetrads or in irregular clusters.
3.2
Primary Isolation Media
Blood agar incubated in 5 - 10% CO2 at 35°C - 37°C for 16 - 24hr.
These organisms may be isolated from other media including CLED, Staph/Strep
selective and Mannitol Salt agar (MSA).
3.3
Colonial Appearance
Staphylococcus species usually grow as opaque, 1 – 5mm, smooth colonies; white,
cream or yellow to orange on blood agar. Haemolysis may be detected. They appear
as white or yellow-green, 1 - 2mm colonies on CLED agar.
Note: Small colony variant strains of S. aureus and strains resistant to vancomycin
(VRSA) may require 72hr incubation to become visible.
S. lugdunensis gives a prominent β-haemolysis and a characteristic Eikenella-like
odour after 2 days of incubation on Columbia agar with 5% sheep blood, which,
combined with colony pleomorphism, helps in its initial recognition65.
Note: Avoid smelling or wafting the bacteria on the plates towards you because of
inhalation of spores and becoming contaminated. This is prohibited in the laboratory.
Micrococcus species produce yellow or red-pigmented colonies on blood agar.
Rothia species are round, convex, mucoidy and adhere to the agar. Colonial
morphology varies with species.
3.4 Test Procedures
3.4.1 Biochemical tests
Catalase test (see TP 8 - Catalase Test)
Staphylococcus, Micrococcus and Rothia species are catalase positive.
S. aureus subspecies anaerobius and S. saccharolyticus are catalase negative22.
Coagulase and other tests to detect S. aureus (see TP 10 - Coagulase Test)
Protein A, clumping factor (slide coagulase or latex), thermostable nuclease or tube
coagulase tests may be used. Positive results or suspected erroneous slide tests
(listed above) may be confirmed by a tube coagulase test.
S. aureus, some strains of S. hyicus, S. intermedius, and S. schleiferi subspecies
coagulans are coagulase positive and thermostable nuclease positive.
Other species of staphylococci are coagulase negative and thermostable nuclease
negative or weak positive. S. lugdunensis is coagulase negative but some strains may
be slide coagulase or clumping factor positive.
Bacteriology – Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 18 of 32
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
Identification of Staphylococcus species, Micrococcus species and Rothia species
DNAse test (see TP 12 – Deoxyribonuclease Test)
Commercially available DNA containing agars are used to detect thermolabile
nuclease activity. Addition of a weak acid (1N HCl) solution to an 18 – 24hr culture
plate will demonstrate clearing around colonies of DNAse positive species and if
toluidine blue O solution is added, a bright rose-pink zone around colonies of DNAse
positive species can be seen.
Note: It is important that both positive and negative screening tests for S. aureus are
verified using a second confirmatory test to detect false positive and false negative
primary screens eg Protein A latex and DNAse.
3.4.2 Commercial identification Systems
Several commercial identification systems are available for the speciation of
Staphylococci. Results should be interpreted in conjunction with the key test results
indicated above.
3.4.3 Matrix-Assisted Laser Desorption/Ionisation - Time of Flight
(MALDI-TOF)
Matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDITOF MS), which can be used to analyse the protein composition of a bacterial cell,
has emerged as a new technology for species identification. This has been shown to
be a rapid and powerful tool because of its reproducibility, speed and sensitivity of
analysis. The advantage of MALDI-TOF as compared with other identification methods
is that the results of the analysis are available within a few hours rather than several
days. The speed and the simplicity of sample preparation and result acquisition
associated with minimal consumable costs make this method well suited for routine
and high-throughput use66.
MALDI-TOF MS method has been found to be useful as an additional test for the
description of new staphylococcal species and in the profiling of staphylococcal strains
and it has also revealed different clonal lineages of S. epidermidis that were of either
human or environmental origin67. However, further studies are required to test this
technology with a large collection of staphylococci of diverse origins.
3.4.4 Nucleic Acid Amplification Tests (NAATs)
PCR is usually considered to be a good method for bacterial detection as it is simple,
sensitive and specific. However, it does have limitations. Although the 16S rRNA gene
is generally targeted for the design of species-specific PCR primers for identification,
designing primers is difficult when the sequences of the homologous genes have high
similarity. In the case of staphylococci, therefore, ribotyping, internal transcribed
spacer PCR and various other methods have been used. There are different PCRs for
the different groups (coagulase positive and the coagulase negative staphylococcal
species) and their target genes and depending on clinical details, the appropriate PCR
will be performed68,69. However, the development of a species-specific quantitative
PCR methodology has proved difficult.
Multiplex PCR assay has also been used for detection of genes encoding surface
protein adhesins, toxins or antibiotic resistance in Staphylococci and more recently, for
species identification of coagulase positive Staphylococci by targeting the
thermonuclease (nuc) gene locus70-72. This has also been used to discriminate
simultaneously between mecA and mecALGA251 alongside the detection of Panton–
Valentine leucocidin (PVL) and nuc genes of Meticillin resistant S. aureus and it
Bacteriology – Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 19 of 32
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
Identification of Staphylococcus species, Micrococcus species and Rothia species
provides a valuable tool for the rapid and accurate characterization of staphylococci
which is essential in modern hospital practice73. This approach would also be valuable
for surveys.
3.5
Further Identification
Toxin studies
Occasionally S. aureus is recovered from cases of suspected toxin-mediated disease
eg staphylococcal scalded skin syndrome, toxic shock syndrome, PVL toxin,
necrotising pneumonia, bullous impetigo and food poisoning. The Staphylococcus
Reference Laboratory welcomes such isolates being referred for toxin gene profiling
and typing studies.
Rapid Methods
A variety of current rapid typing methods have been developed for isolates from
clinical samples; these include molecular techniques such as Pulsed- Field Gel
Electrophoresis (PFGE), 16S rRNA gene sequencing, PCR- restriction fragment
length Polymorphism (PCR-RFLP), spa typing, Multiple-Locus Variable Number
Tandem Repeat Analysis (MLVA) and Multi-locus sequence typing (MLST). All of
these approaches enable subtyping of strains, but do so with different accuracy,
discriminatory power, and reproducibility.
For further molecular investigation, Microarray analysis, single nucleotide
polymorphism (SNPs) – these are looking to replace PFGE and MLVA) and even
whole-genome sequencing (WGS) may be options, as new technology (eg Ion Torrent
Sequencing) allows WGS results within a few days36.
However, some of these methods remain accessible to reference laboratories only
and are difficult to implement for routine bacterial identification in a clinical laboratory.
16S rRNA gene sequencing
16S rRNA gene sequences has been useful in phylogenetic studies at the genus
level, its use has been questioned in studies at the Staphylococcus species level18.
This stems from the fact that closely related species may have identical 16S rRNA
sequences or, alternatively, that divergent 16S rRNA sequences may exist within a
single organism74.
S. caprae and S. capitis cannot be distinguished by their 16S rRNA gene sequences.
Similarly, some Staphylococcus taxa have the same 16S rRNA gene sequences in
variable regions V1, V3, V7, and V9, with identical sequences occurring in, eg
S. vitulinus, S. saccharolyticus, S. capitis subsp urealyticus, S. caprae, the two
subspecies of S. aureus, and the two subspecies of S. cohnii75.
PCR- restriction fragment length Polymorphism (PCR-RFLP)
Due to the limited number of stable features that can be used for species
discrimination, many taxa remain difficult to distinguish from one another and are
misidentified by phenotypic tests.
However, restriction fragment length polymorphism (RFLP) analysis of the dnaJ gene
of PCR products has been reported for use for the identification of staphylococci. This
has proved to be an adequate tool for the correct identification of almost all prevalent
species and subspecies of Staphylococcus, irrespective of their phenotypic
characterization. This method requires only PCR and one or two enzymes and thus is
Bacteriology – Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 20 of 32
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
Identification of Staphylococcus species, Micrococcus species and Rothia species
technically less demanding than the majority of other molecular approaches. It is
easier to use, less expensive and less equipment dependent than sequencing.
This method is also able to discriminate subspecies of the species S. capitis,
S. carnosus, S. cohnii, and S. hominis74.
Pulsed Field Gel Electrophoresis (PFGE)
PFGE detects genetic variation between strains using rare-cutting restriction
endonucleases, followed by separation of the resulting large genomic fragments on an
agarose gel. PFGE is known to be highly discriminatory and a frequently used
technique for outbreak investigations. However, the stability of PFGE may be
insufficient for reliable application in long-term epidemiological studies. Due to its timeconsuming nature (30hr or longer to perform) and its requirement for special
equipment and the interpretation of its results often being subjective, PFGE is not
used widely outside reference laboratories. These problems make the exchange of
strain typing information difficult and complicate the creation of an S. aureus and
MRSA typing database76.
Presently, pulsed-field gel electrophoresis (PFGE) remains the most discriminatory
technique for S. aureus typing, but it allows the constitution of shared databases only
at the national level and is not appropriate for population studies77.
More recently, PFGE has been used for epidemiological typing of Meticillin resistant
Staphylococcus aureus (MRSA)78.
Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA)
Multiple-Locus Variable number tandem repeat Analysis (MLVA) is a method used to
perform molecular typing of particular microorganisms. It utilizes the naturally
occurring variation in the number of tandem repeated DNA sequences found in many
different loci in the genome of a variety of organisms. The molecular typing profiles are
used to study transmission routes, to assess sources of infection and also to assess
the impact of human intervention such as vaccination and use of antibiotics on the
composition of bacterial populations.
This has been used successfully for the genotyping of S. aureus77.
spa sequence typing
spa sequencing appears to be a highly effective rapid typing tool for S. aureus that,
despite some expense of specificity, has significant advantages in terms of speed,
ease of use, ease of interpretation, and standardization among laboratories. It
provides a suitable discrimination for outbreak investigation. Another technique that
can be used in outbreaks is SCCmec typing depending on the local epidemiology
although better SCCmec methods need to be developed36. An additional advantage of
spa typing is that adequate typing information is obtained from a single locus, as
opposed to MLST, which requires the combination of allelic information from many
genes.
It has been documented that spa repeat sequences by themselves define excellent
resolving power among strains of S. aureus79,80. spa typing correctly assigns
staphylococcal strains to the appropriate phylogenetic groups and performs better
than multi locus enzyme electrophoresis (MLEE) and PFGE, and it facilitates the
detection of both macro- and micro-variation81.
Bacteriology – Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 21 of 32
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
Identification of Staphylococcus species, Micrococcus species and Rothia species
However, spa does have some disadvantages. It is insufficiently discriminatory in
regions where a particular clone/small number of clones are endemic; it is not
recommended for smaller local hospital laboratories and is currently not yet used
universally36.
Multi-locus sequence typing (MLST)
MLST measures the DNA sequence variations in a set of housekeeping genes directly
and characterizes strains by their unique allelic profiles. The principle of MLST is
simple: the technique involves PCR amplification followed by DNA sequencing.
Nucleotide differences between strains can be checked at a variable number of genes
depending on the degree of discrimination desired.
This has been used to provide a reliable method of characterising MRSA clones as
well as investigating the epidemiology and phylogeny of S. lugdunensis82. It has also
been used for analysing the evolution of S. epidermidis as well.
Microarrays
DNA microarray technology can provide detailed, clinically relevant information on the
isolate by detecting the presence or absence of a large number of virulenceassociated genes simultaneously in a single assay; however, their clinical value has
been limited by a complicated methodology that is unsuitable for routine use in
diagnostic microbiology laboratories.
This has been used to successfully differentiate between isolates representative of a
spectrum of S. aureus types, including meticillin susceptible, meticillin resistant,
community-acquired, and vancomycin resistant S. aureus, and to simultaneously
detect clinically relevant virulence determinants83.
Whole Genome Sequencing (WGS)
This is also known as full genome sequencing, complete genome sequencing, or
entire genome sequencing. It is a laboratory process that determines the complete
DNA sequence of an organism's genome at a single time. There are several highthroughput techniques that are available and used to sequence an entire genome
such as pyrosequencing, nanopore technology, IIIumina sequencing, Ion Torrent
sequencing, etc. This sequencing method holds great promise for rapid, accurate, and
comprehensive identification of bacterial transmission pathways in hospital and
community settings, with concomitant reductions in infections, morbidity, and costs84.
This has been useful in the detection of meticillin resistant S. aureus in an outbreak85.
It has also been used to highlight extensive differences in genome content between
the closely related Staphylococcus intermedius group (S. intermedius,
S. pseudintermedius and S. delphini) inhabiting distinct host niches as well as
providing new avenues for research into pathogenesis and bacterial host-adaptation13.
3.6
Storage and Referral
If required, save pure isolate on a nutrient agar slope for referral to the Reference
Laboratory.
Any strain of S. aureus suspected of demonstrating unusual resistance eg vancomyin,
linezolid must be referred to the Staphylococcal Reference Service for further
examination.
Bacteriology – Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 22 of 32
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
Identification of Staphylococcus species, Micrococcus species and Rothia species
4 Identification of Staphylococcus Species,
Micrococcus species and Rothia species
Clinical specimens
Primary isolation plate
Opaque, white, cream, yellow or orange colonies on blood agar
Gram stain
Gram positive cocci in clusters
If there is a different Gram stain
appearance refer to the appropriate UKSMI
Suspected
S. aureus
Catalase
Positive
Negative
Catalase negative
S. aureus subsp anaerobius S.capitis
S. saccharolyticus
(grows anaerobically)
Rothia dentocariosa*
Consider other organisms
Catalase positive
All Staphylococcus,
Micrococcus and Rothia
species
Novobiocin
sensitivity test
Resistant
S. saprophyticus,
S. sciuri and
S. cohnii groups
S. hominis subsp.
novobiosepticus
S. cohnii group
Sensitive
All Staphylococcus species
Coagulase (slide or tube)
DNAse test, Commercial
identification system
Negative
Coagulase-negative
Staphylococcus species
Positive
S. aureus
S. hyicus*
S. intermedius*
S. schleiferi subsp coagulans*
Confirm with tube
coagulase if required
Further identification if clinically indicated
Refer to the Reference Laboratory
If required, save the pure isolate
onto a nutrient agar slope
* Strains may be positive to certain tests
Bacteriology – Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 23 of 32
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
Identification of Staphylococcus species, Micrococcus species and Rothia species
5
Reporting
5.1
Presumptive Identification
If appropriate growth characteristics, colonial appearance, Gram stain of the culture,
catalase and slide coagulase or latex agglutination results are demonstrated.
5.2
Confirmation of Identification
Following confirmatory test results.
5.3
Medical Microbiologist
Inform the medical microbiologist of presumptive and confirmed Staphylococcus
aureus when the request bears relevant information, eg:
•
Toxin-mediated phenomena (eg Toxic Shock Syndrome, scalded skin
syndrome, epidermal necrolysis, bullous impetigo, necrotising pneumonia, food
poisoning)
•
Suspected outbreaks or instances of cross-infection
The medical microbiologist should also be informed of presumptive and confirmed
isolates of Staphylococcus species under the following circumstances:
•
Osteomyelitis and septic arthritis
•
Infections involving indwelling medical devices, eg prosthetic valves,
pacemakers, CSF shunts, peritoneal or vascular catheters
•
Endocarditis, haematogenous dissemination of infection, septicaemia
•
Isolates from normally sterile sites
•
Serious soft-tissue infections (cellulitis, erysipelas, necrotising myofasciitis,
puerperal sepsis, surgical wound infection, pneumonia, peritonitis, meningitis,
formation of abscesses or empyemas)
Follow local protocols for reporting to clinician.
5.4
CCDC
Refer to local Memorandum of Understanding.
5.5
Public Health England86
Refer to current guidelines on CIDSC and COSURV reporting.
5.6
Infection Prevention and Control Team
Inform the infection prevention and control team of isolates of meticillin resistant
Staphylococcus aureus and any S. aureus bacteremia (MSSA) in accordance with
local protocols.
Bacteriology – Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 24 of 32
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
Identification of Staphylococcus species, Micrococcus species and Rothia species
6
Referrals
6.1
Reference Laboratory
Contact appropriate devolved national reference laboratory for information on the tests
available, turnaround times, transport procedure and any other requirements for
sample submission:
Staphylococcus Reference Service
Antimicrobial Resistance and Healthcare Associated Infections Reference Unit
Microbiology Services
Public Health England
61 Colindale Avenue
London
NW9 5EQ
Contact PHE’s main switchboard: Tel. +44 (0) 20 8200 4400
England and Wales
https://www.gov.uk/specialist-and-reference-microbiology-laboratory-tests-andservices
Scotland
http://www.hps.scot.nhs.uk/reflab/index.aspx
Northern Ireland
http://www.belfasttrust.hscni.net/Laboratory-MortuaryServices.htm
7 Notification to PHE86,87 or Equivalent in the
Devolved Administrations88-91
The Health Protection (Notification) regulations 2010 require diagnostic laboratories to
notify Public Health England (PHE) when they identify the causative agents that are
listed in Schedule 2 of the Regulations. Notifications must be provided in writing, on
paper or electronically, within seven days. Urgent cases should be notified orally and
as soon as possible, recommended within 24 hours. These should be followed up by
written notification within seven days.
For the purposes of the Notification Regulations, the recipient of laboratory
notifications is the local PHE Health Protection Team. If a case has already been
notified by a registered medical practitioner, the diagnostic laboratory is still required
to notify the case if they identify any evidence of an infection caused by a notifiable
causative agent.
Notification under the Health Protection (Notification) Regulations 2010 does not
replace voluntary reporting to PHE. The vast majority of NHS laboratories voluntarily
report a wide range of laboratory diagnoses of causative agents to PHE and many
PHE Health protection Teams have agreements with local laboratories for urgent
reporting of some infections. This should continue.
Note: The Health Protection Legislation Guidance (2010) includes reporting of Human
Immunodeficiency Virus (HIV) & Sexually Transmitted Infections (STIs), Healthcare
Associated Infections (HCAIs) and Creutzfeldt–Jakob disease (CJD) under
Bacteriology – Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 25 of 32
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
Identification of Staphylococcus species, Micrococcus species and Rothia species
‘Notification Duties of Registered Medical Practitioners’: it is not noted under
‘Notification Duties of Diagnostic Laboratories’.
https://www.gov.uk/government/organisations/public-health-england/about/ourgovernance#health-protection-regulations-2010
Other arrangements exist in Scotland88,89, Wales90 and Northern Ireland91.
Bacteriology – Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 26 of 32
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
Identification of Staphylococcus species, Micrococcus species and Rothia species
References
1.
Prax M, Lee CY, Bertram R. An update on the molecular genetics toolbox for staphylococci.
Microbiology 2013;159:421-35.
2.
Kloos WE, Bannerman TL. Update on clinical significance of coagulase-negative staphylococci.
Clin Microbiol Rev 1994;7:117-40.
3.
Euzeby,JP. List of Prokaryotic names with standing in Nomenclature- Genus Micrococcus. 2013.
4.
von GA. Rothia dentocariosa: taxonomy and differential diagnosis. Clin Microbiol Infect
2004;10:399-402.
5.
Euzeby,JP. List of prokaryotic names with standing in Nomenclature- Genus Rothia. 2013.
6.
Webster JA, Bannerman TL, Hubner RJ, Ballard DN, Cole EM, Bruce JL, et al. Identification of
the Staphylococcus sciuri species group with EcoRI fragments containing rRNA sequences and
description of Staphylococcus vitulus sp. nov. Int J Syst Bacteriol 1994;44:454-60.
7.
Vernozy-Rozand C, Mazuy C, Meugnier H, Bes M, Lasne Y, Fiedler F, et al. Staphylococcus
fleurettii sp. nov., isolated from goat's milk cheeses. Int J Syst Evol Microbiol 2000;50 Pt 4:15217.
8.
Holt JG. Gram-Positive cocci. In: Holt JG, Krieg NR, Sneath PHA, Staley JT, Williams ST, editors.
Bergey's Manual of Determinative Bacteriology. 9th ed. Baltimore; 1994. p. 527-37.
9.
Fontana C, Cellini L, Dainelli B. Twelve aberrant strains of Staphylococcus aureus subsp. aureus
from clinical specimens. J Clin Microbiol 1993;31:2105-9.
10.
Fuente De LA R, Suarez G, Schleifer KH. Staphylococcus aureus subsp. anaerobius subsp. nov.,
the causal agent of abscess disease of sheep. International Journal of Systematic Bacteriology
1985;35:99-102.
11.
Vandenesch F, Lebeau C, Bes M, McDevitt D, Greenland T, Novick RP, et al. Coagulase
deficiency in clinical isolates of Staphylococcus aureus involves both transcriptional and posttranscriptional defects. J Med Microbiol 1994;40:344-9.
12.
Devriese LA, Vancanneyt M, Baele M, Vaneechoutte M, De GE, Snauwaert C, et al.
Staphylococcus pseudintermedius sp. nov., a coagulase-positive species from animals. Int J Syst
Evol Microbiol 2005;55:1569-73.
13.
Ben Zakour NL, Beatson SA, van den Broek AH, Thoday KL, Fitzgerald JR. Comparative
genomics of the Staphylococcus intermedius group of animal pathogens. Front Cell Infect
Microbiol 2012;2:44.
14.
HALE JH. Studies on staphylococcus mutation: a naturally occurring "G" gonidial variant and its
carbon dioxide requirements. Br J Exp Pathol 1951;32:307-13.
15.
Proctor RA, von Eiff C, Kahl BC, Becker K, McNamara P, Herrmann M, et al. Small colony
variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections. Nat Rev
Microbiol 2006;4:295-305.
16.
Foster. Staphylococcus Aureus. Molecular Medical Microbiology. 2002. p. 839-88.
17.
Rupp ME, Archer GL. Coagulase-negative staphylococci: pathogens associated with medical
progress. Clin Infect Dis 1994;19:231-43.
Bacteriology – Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 27 of 32
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
Identification of Staphylococcus species, Micrococcus species and Rothia species
18.
Takahashi T, Satoh I, Kikuchi N. Phylogenetic relationships of 38 taxa of the genus
Staphylococcus based on 16S rRNA gene sequence analysis. Int J Syst Bacteriol 1999;49 Pt
2:725-8.
19.
Christensen GD, Parisi JT, Bisno AL, Simpson WA, Beachey EH. Characterization of clinically
significant strains of coagulase-negative staphylococci. J Clin Microbiol 1983;18:258-69.
20.
Jansen B, Schumacher-Perdreau F, Peters G, Pulverer G. New aspects in the pathogenesis and
prevention of polymer-associated foreign-body infections caused by coagulase-negative
staphylococci. J Invest Surg 1989;2:361-80.
21.
Koneman EW, Allen SD, Janda WM, et al. Gram-Positive Cocci Part 1: Staphylococci and
Related Gram-Positive Cocci. Koneman's Colour Atlas and Textbook of Diagnostic Microbiology.
6th ed. Philadelphia: Lippincott Williams & Wilkins; 2006. p. 623-71.
22.
Kloos W, Bannerman TL. Staphylococcus and Micrococcus. In: Murray PR, Baron EJ, Pfaller MA,
Tenover FC, Yolken RH, editors. Manual of Clinical Microbiology. 7th ed. Washington DC:
American Society for Microbiology; 1999. p. 264-82.
23.
Vandenesch F, Perrier-Gros-Claude JD, Bes M, Fuhrmann C, Delorme V, Mouren C, et al.
Staphylococcus pasteuri-specific oligonucleotide probes derived from a random amplified DNA
fragment
259. FEMS Microbiol Lett 1995;132:147-52.
24.
Schleifer KH, Kloos WE. Isolation and Characterization of Staphylococci from Human Skin.
International Journal of Systematic Bacteriology 1975;25:50-61.
25.
Hajek V, Meugnier H, Bes M, Brun Y, Fiedler F, Chmela Z, et al. Staphylococcus saprophyticus
subsp. bovis subsp. nov., isolated from bovine nostrils. Int J Syst Bacteriol 1996;46:792-6.
26.
Baker JS. Comparison of various methods for differentiation of staphylococci and micrococci. J
Clin Microbiol 1984;19:875-9.
27.
Faller A, Schleifer KH. Modified oxidase and benzidine tests for separation of staphylococci from
micrococci. J Clin Microbiol 1981;13:1031-5.
28.
Wieser M, Denner EB, Kampfer P, Schumann P, Tindall B, Steiner U, et al. Emended
descriptions of the genus Micrococcus, Micrococcus luteus (Cohn 1872) and Micrococcus lylae
(Kloos et al. 1974). Int J Syst Evol Microbiol 2002;52:629-37.
29.
Kloos WE, Tornabene TG, Schleifer KH. Isolation and characterization of Micrococci from human
skin, including two new species: Micrococus lylae and Micrococcus kristinae. International
Journal of Systematic Bacteriology 1974;24:79-101.
30.
Georg LK, Brown JM. Rothia, Gen. Nov. an aerobic genus of the family actinomycetaceae.
International Journal of Systematic Bacteriology 1967;17:79-88.
31.
Funke G, von Graevenitz A, Clarridge JE, III, Bernard KA. Clinical microbiology of coryneform
bacteria. Clin Microbiol Rev 1997;10:125-59.
32.
van Tiel FH, Slangen BF, Schouten HC, Jacobs JA. Study of Stomatococcus mucilaginosus
isolated in a hospital ward using phenotypic characterization. Eur J Clin Microbiol Infect Dis
1995;14:193-8.
33.
Collins MD, Hutson RA, Baverud V, Falsen E. Characterization of a Rothia-like organism from a
mouse: description of Rothia nasimurium sp. nov. and reclassification of Stomatococcus
mucilaginosus as Rothia mucilaginosa comb. nov. Int J Syst Evol Microbiol 2000;50 Pt 3:1247-51.
Bacteriology – Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 28 of 32
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
Identification of Staphylococcus species, Micrococcus species and Rothia species
34.
Ruoff KL. Miscellaneous catalase-negative, gram-positive cocci: emerging opportunists. J Clin
Microbiol 2002;40:1129-33.
35.
Personne P, Bes M, Lina G, Vandenesch F, Brun Y, Etienne J. Comparative performances of six
agglutination kits assessed by using typical and atypical strains of Staphylococcus aureus. J Clin
Microbiol 1997;35:1138-40.
36.
Stefani S, Chung DR, Lindsay JA, Friedrich AW, Kearns AM, Westh H, et al. Meticillin-resistant
Staphylococcus aureus (MRSA): global epidemiology and harmonisation of typing methods. Int J
Antimicrob Agents 2012;39:273-82.
37.
Perry JD, Rennison C, Butterworth LA, Hopley AL, Gould FK. Evaluation of S. aureus ID, a new
chromogenic agar medium for detection of Staphylococcus aureus. J Clin Microbiol
2003;41:5695-8.
38.
Carricajo A, Treny A, Fonsale N, Bes M, Reverdy ME, Gille Y, et al. Performance of the
chromogenic medium CHROMagar Staph Aureus and the Staphychrom coagulase test in the
detection and identification of Staphylococcus aureus in clinical specimens. J Clin Microbiol
2001;39:2581-3.
39.
Kluytmans J, Van GA, Willemse P, Van KP. Performance of CHROMagar selective medium and
oxacillin resistance screening agar base for identifying Staphylococcus aureus and detecting
methicillin resistance. J Clin Microbiol 2002;40:2480-2.
40.
Brown DF, Edwards Di, Hawkey PM, Morrison D, Ridgway GL, Towner KJ, et al. Guidelines for
the laboratory diagnosis and susceptibility testing of methicillin-resistant Staphylococcus aureus
(MRSA). J Antimicrob Chemother 2005;56:1000-18.
41.
Yrios JW. Comparison of rabbit and pig plasma in the tube coagulase test. J Clin Microbiol
1977;5:221-4.
42.
European Parliament. UK Standards for Microbiology Investigations (SMIs) use the term "CE
marked leak proof container" to describe containers bearing the CE marking used for the
collection and transport of clinical specimens. The requirements for specimen containers are
given in the EU in vitro Diagnostic Medical Devices Directive (98/79/EC Annex 1 B 2.1) which
states: "The design must allow easy handling and, where necessary, reduce as far as possible
contamination of, and leakage from, the device during use and, in the case of specimen
receptacles, the risk of contamination of the specimen. The manufacturing processes must be
appropriate for these purposes".
43.
Official Journal of the European Communities. Directive 98/79/EC of the European Parliament
and of the Council of 27 October 1998 on in vitro diagnostic medical devices. 7-12-1998. p. 1-37.
44.
Health and Safety Executive. Safe use of pneumatic air tube transport systems for pathology
specimens. 9/99.
45.
Department for transport. Transport of Infectious Substances, 2011 Revision 5. 2011.
46.
World Health Organization. Guidance on regulations for the Transport of Infectious Substances
2013-2014. 2012.
47.
Home Office. Anti-terrorism, Crime and Security Act. 2001 (as amended).
48.
Advisory Committee on Dangerous Pathogens. The Approved List of Biological Agents. Health
and Safety Executive. 2013. p. 1-32
49.
Advisory Committee on Dangerous Pathogens. Infections at work: Controlling the risks. Her
Majesty's Stationery Office. 2003.
Bacteriology – Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 29 of 32
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
Identification of Staphylococcus species, Micrococcus species and Rothia species
50.
Advisory Committee on Dangerous Pathogens. Biological agents: Managing the risks in
laboratories and healthcare premises. Health and Safety Executive. 2005.
51.
Advisory Committee on Dangerous Pathogens. Biological Agents: Managing the Risks in
Laboratories and Healthcare Premises. Appendix 1.2 Transport of Infectious Substances Revision. Health and Safety Executive. 2008.
52.
Centers for Disease Control and Prevention. Guidelines for Safe Work Practices in Human and
Animal Medical Diagnostic Laboratories. MMWR Surveill Summ 2012;61:1-102.
53.
Health and Safety Executive. Control of Substances Hazardous to Health Regulations. The
Control of Substances Hazardous to Health Regulations 2002. 5th ed. HSE Books; 2002.
54.
Health and Safety Executive. Five Steps to Risk Assessment: A Step by Step Guide to a Safer
and Healthier Workplace. HSE Books. 2002.
55.
Health and Safety Executive. A Guide to Risk Assessment Requirements: Common Provisions in
Health and Safety Law. HSE Books. 2002.
56.
Health Services Advisory Committee. Safe Working and the Prevention of Infection in Clinical
Laboratories and Similar Facilities. HSE Books. 2003.
57.
British Standards Institution (BSI). BS EN12469 - Biotechnology - performance criteria for
microbiological safety cabinets. 2000.
58.
British Standards Institution (BSI). BS 5726:2005 - Microbiological safety cabinets. Information to
be supplied by the purchaser and to the vendor and to the installer, and siting and use of
cabinets. Recommendations and guidance. 24-3-2005. p. 1-14
59.
Savini V, Barbarini D, Polakowska K, Gherardi G, Bialecka A, Kasprowicz A, et al. Methicillinresistant Staphylococcus pseudintermedius infection in a bone marrow transplant recipient. J Clin
Microbiol 2013;51:1636-8.
60.
Al MM, Raoult D, Roux V. Staphylococcus massiliensis sp. nov., isolated from a human brain
abscess. Int J Syst Evol Microbiol 2010;60:1066-72.
61.
Stepanovic S, Jezek P, Dakic I, Vukovic D, Seifert L. Staphylococcus sciuri: an unusual cause of
pelvic inflammatory disease. Int J STD AIDS 2005;16:452-3.
62.
Dakic I, Morrison D, Vukovic D, Savic B, Shittu A, Jezek P, et al. Isolation and molecular
characterization of Staphylococcus sciuri in the hospital environment. J Clin Microbiol
2005;43:2782-5.
63.
Stepanovic S, Dakic I, Morrison D, Hauschild T, Jezek P, Petras P, et al. Identification and
characterization of clinical isolates of members of the Staphylococcus sciuri group. J Clin
Microbiol 2005;43:956-8.
64.
Vandenesch F, Lebeau C, Bes M, Lina G, Lina B, Greenland T, et al. Clotting activity in
Staphylococcus schleiferi subspecies from human patients. J Clin Microbiol 1994;32:388-92.
65.
Bocher S, Tonning B, Skov RL, Prag J. Staphylococcus lugdunensis, a common cause of skin
and soft tissue infections in the community. J Clin Microbiol 2009;47:946-50.
66.
Barbuddhe SB, Maier T, Schwarz G, Kostrzewa M, Hof H, Domann E, et al. Rapid identification
and typing of listeria species by matrix-assisted laser desorption ionization-time of flight mass
spectrometry. Appl Environ Microbiol 2008;74:5402-7.
Bacteriology – Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 30 of 32
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
Identification of Staphylococcus species, Micrococcus species and Rothia species
67.
Dubois D, Leyssene D, Chacornac JP, Kostrzewa M, Schmit PO, Talon R, et al. Identification of a
variety of Staphylococcus species by matrix-assisted laser desorption ionization-time of flight
mass spectrometry. J Clin Microbiol 2010;48:941-5.
68.
Gribaldo S, Cookson B, Saunders N, Marples R, Stanley J. Rapid identification by specific PCR
of coagulase-negative staphylococcal species important in hospital infection. J Med Microbiol
1997;46:45-53.
69.
Edwards KJ, Kaufmann ME, Saunders NA. Rapid and accurate identification of coagulasenegative staphylococci by real-time PCR. J Clin Microbiol 2001;39:3047-51.
70.
Stepan J, Pantucek R, Doskar J. Molecular diagnostics of clinically important staphylococci. Folia
Microbiol (Praha) 2004;49:353-86.
71.
Sasaki T, Tsubakishita S, Tanaka Y, Sakusabe A, Ohtsuka M, Hirotaki S, et al. Multiplex-PCR
method for species identification of coagulase-positive staphylococci. J Clin Microbiol
2010;48:765-9.
72.
Hirotaki S, Sasaki T, Kuwahara-Arai K, Hiramatsu K. Rapid and accurate identification of humanassociated staphylococci by use of multiplex PCR. J Clin Microbiol 2011;49:3627-31.
73.
Pichon B, Hill R, Laurent F, Larsen AR, Skov RL, Holmes M, et al. Development of a real-time
quadruplex PCR assay for simultaneous detection of nuc, Panton-Valentine leucocidin (PVL),
mecA and homologue mecALGA251. J Antimicrob Chemother 2012;67:2338-41.
74.
Hauschild T, Stepanovic S. Identification of Staphylococcus spp. by PCR-restriction fragment
length polymorphism analysis of dnaJ gene. J Clin Microbiol 2008;46:3875-9.
75.
Ghebremedhin B, Layer F, Konig W, Konig B. Genetic classification and distinguishing of
Staphylococcus species based on different partial gap, 16S rRNA, hsp60, rpoB, sodA, and tuf
gene sequences. J Clin Microbiol 2008;46:1019-25.
76.
Tenover FC, Arbeit R, Archer G, Biddle J, Byrne S, Goering R, et al. Comparison of traditional
and molecular methods of typing isolates of Staphylococcus aureus. J Clin Microbiol
1994;32:407-15.
77.
Pourcel C, Hormigos K, Onteniente L, Sakwinska O, Deurenberg RH, Vergnaud G. Improved
multiple-locus variable-number tandem-repeat assay for Staphylococcus aureus genotyping,
providing a highly informative technique together with strong phylogenetic value. J Clin Microbiol
2009;47:3121-8.
78.
Murchan S, Kaufmann ME, Deplano A, de RR, Struelens M, Zinn CE, et al. Harmonization of
pulsed-field gel electrophoresis protocols for epidemiological typing of strains of methicillinresistant Staphylococcus aureus: a single approach developed by consensus in 10 European
laboratories and its application for tracing the spread of related strains. J Clin Microbiol
2003;41:1574-85.
79.
Shopsin B, Gomez M, Montgomery SO, Smith DH, Waddington M, Dodge DE, et al. Evaluation of
protein A gene polymorphic region DNA sequencing for typing of Staphylococcus aureus strains.
J Clin Microbiol 1999;37:3556-63.
80.
Harmsen D, Claus H, Witte W, Rothganger J, Claus H, Turnwald D, et al. Typing of methicillinresistant Staphylococcus aureus in a university hospital setting by using novel software for spa
repeat determination and database management. J Clin Microbiol 2003;41:5442-8.
81.
Koreen L, Ramaswamy SV, Graviss EA, Naidich S, Musser JM, Kreiswirth BN. spa typing method
for discriminating among Staphylococcus aureus isolates: implications for use of a single marker
to detect genetic micro- and macrovariation. J Clin Microbiol 2004;42:792-9.
Bacteriology – Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 31 of 32
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
Identification of Staphylococcus species, Micrococcus species and Rothia species
82.
Chassain B, Lemee L, Didi J, Thiberge JM, Brisse S, Pons JL, et al. Multilocus sequence typing
analysis of Staphylococcus lugdunensis implies a clonal population structure. J Clin Microbiol
2012;50:3003-9.
83.
Spence RP, Wright V, Ala-Aldeen DA, Turner DP, Wooldridge KG, James R. Validation of
virulence and epidemiology DNA microarray for identification and characterization of
Staphylococcus aureus isolates. J Clin Microbiol 2008;46:1620-7.
84.
Koser CU, Ellington MJ, Cartwright EJ, Gillespie SH, Brown NM, Farrington M, et al. Routine use
of microbial whole genome sequencing in diagnostic and public health microbiology. PLoS
Pathog 2012;8:e1002824.
85.
Harris SR, Cartwright EJ, Torok ME, Holden MT, Brown NM, Ogilvy-Stuart AL, et al. Wholegenome sequencing for analysis of an outbreak of meticillin-resistant Staphylococcus aureus: a
descriptive study. Lancet Infect Dis 2013;13:130-6.
86.
Public Health England. Laboratory Reporting to Public Health England: A Guide for Diagnostic
Laboratories. 2013. p. 1-37.
87.
Department of Health. Health Protection Legislation (England) Guidance. 2010. p. 1-112.
88.
Scottish Government. Public Health (Scotland) Act. 2008 (as amended).
89.
Scottish Government. Public Health etc. (Scotland) Act 2008. Implementation of Part 2: Notifiable
Diseases, Organisms and Health Risk States. 2009.
90.
The Welsh Assembly Government. Health Protection Legislation (Wales) Guidance. 2010.
91.
Home Office. Public Health Act (Northern Ireland) 1967 Chapter 36. 1967 (as amended).
Bacteriology – Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 32 of 32
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England